Molecular phylogenetics at the population/species interface in cave spiders of the southern Appalachians (Araneae:Nesticidae:Nesticus)

This paper focuses on the relationship between population genetic structure and speciation mechanisms in a monophyletic species group of Appalachian cave spiders (Nesticus). Using mtDNA sequence data gathered from 256 individuals, I analyzed patterns of genetic variation within and between populations for three pairs of closely related sister species. Each sister-pair comparison involves taxa with differing distributional and ecological attributes; if these ecological attributes are reflected in basic demographic differences, then speciation might proceed differently across these sister taxa comparisons. Both frequency-based and gene tree analyses reveal that the genetic structure of the Nesticus species studied is characterized by similar and essentially complete population subdivision, regardless of differences in general ecology. These findings contrast with results of prior genetic studies of cave-dwelling arthropods that have typically revealed variation in population structure corresponding to differences in general ecology. Species fragmentation through both extrinsic and intrinsic evolutionary forces has resulted in discrete, perhaps independent, populations within morphologically defined species. Large sequence divergence values observed between populations suggest that this independence may extend well into the past. These patterns of mtDNA genealogical structure and divergence imply that species as morphological lineages are currently more inclusive than basal evolutionary or phylogenetic units, a suggestion that has important implications for the study of speciation mechanisms.      

Contribution of S-Layer Proteins to the Mosquitocidal Activity of Lysinibacillus sphaericus

Lysinibacillus sphaericus strains belonging the antigenic group H5a5b produce spores with larvicidal activity against larvae of Culex mosquitoes. C7, a new isolated strain, which presents similar biochemical characteristics and Bin toxins in their spores as the reference strain 2362, was, however, more active against larvae of Culex mosquitoes. The contribution of the surface layer protein (S-layer) to this behaviour was envisaged since this envelope protein has been implicated in the pathogenicity of several bacilli, and we had previously reported its association to spores. Microscopic observation by immunofluorescence detection with anti S-layer antibody in the spores confirms their attachment. S-layers and BinA and BinB toxins formed high molecular weight multimers in spores as shown by SDS-PAGE and western blot detection. Purified S-layer from both L. sphaericus C7 and 2362 strain cultures was by itself toxic against Culex sp larvae, however, that from C7 strain was also toxic against Aedes aegypti. Synergistic effect between purified S-layer and spore-crystal preparations was observed against Culex sp. and Aedes aegypti larvae. This effect was more evident with the C7 strain. In silico analyses of the S-layer sequence suggest the presence of chitin-binding and hemolytic domains. Both biochemical characteristics were detected for both S-layers strains that must justify their contribution to pathogenicity.     

Trimetazidine improves left ventricular function in diabetic patients with coronary artery disease: a double-blind placebo-controlled study

Background

Patients with diabetic cardiomyopathy have an impaired myocardial glucose handling and distal distribution of coronary atherosclerosis. Trimetazidine, an anti-ischemic metabolic agent, improves myocardial glucose utilization though inhibition of fatty acid oxidation. Aim of the present study was to evaluate whether the metabolic effect of trimetazidine on left ventricular function in patients with diabetic cardiomyopathy.

Methods

32 patients (24 males and 8 females, mean (SE) age = 67 ± 6 years) with type 2 diabetes and ischemic cardiomyopathy were randomized to receive either trimetazidine (20 mg, t.d.s.) or placebo (t.d.s.) for six months in a randomized parallel study. Patients performed an echocardiogram at baseline and after 6 months.

Results

Demographic data were comparable between the two groups. After six month baseline left ventricular end-diastolic diameters increased from 62.4 ± 1.7 to 63 ± 2.1 mm in the placebo group, while decreased from 63.2 ± 2.1 to 58 ± 1.6 mm (p < 0.01 compared to baseline) in the trimetazidine group. Compared to baseline, left ventricular ejection fraction increased by 5.4 ± 0.5% (p < 0.05) in the trimetazidine group while remained unchanged in the placebo group -2.4 ± 1.1% (NS), p < 0.01 between groups. A significant improvement in wall motion score index and in the E/A wave ratio was detected in patients treated with trimetazidine, but not in those receiving placebo.

Conclusion

in diabetic patients with ischemic heart disease trimetazidine added to standard medical therapy has beneficial effect on left ventricular volumes and on left ventricular ejection fraction compared to placebo. This effect may be related to the effect of trimetazidine upon cardiac glucose utilization.     

Development of sequence amplified characterized region (SCAR) markers of helicoverpa armigera: a new polymerase chain reaction-based technique for predator gut analysis

A method is described for the development of DNA markers for detection of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) in predator gut analysis, based on sequence characterized amplified regions (SCARs) derived from a randomly amplified polymorphic DNA (RAPD) band. A 1200-bp DNA fragment of H. armigera, absent in the predator band pattern and in other closely related prey species, was identified by RAPD analysis. This fragment was cloned and its extremes sequenced to design extended strand-specific 20-mer oligonucleotide primers. Three pairs of SCAR primers, which amplified three different DNA fragments, were used to study the effect of fragment length on detection of prey in the predator gut. Using the pair of primers that amplified the longest fragment of H. armigera DNA, a single band of 1100 bp was obtained, but its detection was not possible in the predator gut. Detection of the ingested prey was possible with the other two pairs of SCAR primers, obtaining bands of 600 and 254 bp, respectively. Detection of H. armigera DNA in the gut of the predator Dicyphus tamaninii was evaluated immediately after ingestion (t = 0) and after 4 h. Detection of H. armigera DNA after 4 h was only possible using the pair of primers that amplified the shortest fragment (254 bp). The test for specificity, using these last pair of primers, showed that H. armigera was the only species detected. The detection threshold was defined at a 1:8192 dilution of a H. armigera whole egg in all samples.     

Synergistic Effects of the Lactobacillus acidophilus Surface Layer and Nisin on Bacterial Growth

We have previously described a murein hydrolase activity for the surface layer (S-layer) of Lactobacillus acidophilus ATCC 4356. Here we show that, in combination with nisin, this S-layer acts synergistically to inhibit the growth of pathogenic Gram-negative Salmonella enterica and potential pathogenic Gram-positive bacteria, Staphylococcus aureus and Bacillus cereus. In addition, bacteriolytic effects were observed for the Gram-positive species tested. We postulate that the S-layer enhances the access of nisin into the cell membrane by enabling it to cross the cell wall, while nisin provides the sudden ion-nonspecific dissipation of the proton motive force required to enhance the S-layer murein hydrolase activity.Natural preservatives active against Gram-positive and Gram-negative pathogens are highly desirable for the food industry and consumers. Nisin, a small peptide bacteriocin produced by Lactococcus lactis subsp. lactis, is the only bacteriocin that has approved GRAS (generally recognized as safe) status for certain applications (Generally Regarded as Safe; US 21CFR170.30-Food Additives) by the Food and Drug Administration (FDA) for use in food products (3, 19). The antimicrobial mechanism of nisin has been extensively studied and is well documented (1, 5, 8, 9, 20). Nisin is not active against Gram-negative bacteria compared to Gram-positive bacteria, due to its inability to penetrate the external membrane, which prevents its access to the inner membrane. However, the combination of nisin with the chelating agent lactoferrin acts synergistically to inhibit the growth of Escherichia coli O157:H7 (13). Nisin has been used as a food preservative for over 30 years, is active in the nanomolar range, and has no known toxicity to humans (5, 19), which has placed it in the unique position of worldwide acceptance as a powerful and safe food additive in control of food spoilage and certain food pathogens.We have previously described a new enzymatic functionality for the surface layer (S-layer) of Lactobacillus acidophilus ATCC 4356, namely, an endopeptidase activity against cell wall preparations of Salmonella enterica serovar Newport (17); however, we failed to observe any effect on whole cells from Gram-positive bacteria such as Bacillus cereus.We now wonder if the purified S-layer protein having this endopeptidase activity may act synergistically with nisin, allowing reduction of the levels of the bacteriocin and control of bacterial growth. For this purpose, the antibacterial activity of S-layer from Lactobacillus acidophilus ATCC 4356, either alone or in combination with nisin, was analyzed using three models of food-borne pathogenic bacteria: Salmonella enterica serovar Newport (10), Staphylococcus aureus ATCC 6538 (used as a representative of Gram-positive bacteria in standard medium tests; laboratory collection), and Bacillus cereus 6A1 (Bacillus Genetic Stock Center [BGSC]). The S-layer proteins were extracted as previously described (17) and conserved as a 1-mg/ml suspension in sterile distilled water at −20°C until use. Purity was determined by SDS-PAGE as a unique band and detected by Western blotting.Nisin was used as its commercial product, 2.5% (wt/wt) (Sigma), and was dissolved in 0.02 N HCl to a concentration of 16,000 IU/ml, equivalent to 0.4 mg/ml pure nisin. The MIC that inhibited growth was determined twice by microtiter broth dilution, and we measured the optical density at 600 nm (OD₆₀₀) reached after 16 h of growth. Bacillus cereus and Salmonella enterica were grown in LB (yeast extract, 5 g/liter; peptone, 10 g/liter; NaCl, 10 g/liter; pH 7.0), while Staphylococcus aureus was grown in BHI (brain heart infusion; Biokar, France) and incubated at 37°C. The MIC was 595 IU/ml for Bacillus cereus, 298 IU/ml for Staphylococcus aureus, and over 5,000 IU/ml for Salmonella enterica serovar Newport. For Salmonella enterica, nisin solution was prepared to a concentration of 160,000 IU/ml, equivalent to 4 mg/ml pure nisin, in order to avoid a pH modification once it was added to the growth medium.In a first approach, growth curves of the Gram-negative Salmonella enterica serovar Newport in the presence of nisin, S-layer, or both were performed. Nisin at half of its MIC showed no inhibition. The S-layer alone was inhibitory, but the addition of both nisin and S-layer decreased growth (for growth rate and maximal OD reached, see Fig. ​Fig.1).1). In view of these findings, we decided to evaluate the effects of the combination (nisin and S-layer) in Gram-positive bacteria. Bacillus cereus and Staphylococcus aureus, two food-borne pathogen models, were chosen. No effect was observed when the S-layer (10 μg/ml) was added alone, and nisin added alone, at a subinhibitory concentration of 250 IU/ml for B.cereus and 150 IU/ml for S. aureus, partially delayed growth. In contrast, the combination of both the S-layer (10 μg/ml) and nisin at a subinhibitory concentration inhibited growth of both B. cereus and S. aureus cultures (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Effect of S-layer, nisin, or both on the growth of S. enterica, S. aureus, and B. cereus. To determine the effect of nisin and S-layer protein separately or in combination, bacterial cells were first cultured at 37°C for 16 h with agitation. These cultures were diluted in fresh medium (0.5 ml into 10 ml) and distributed in aliquots, each containing either the S-layer, nisin, or both, and OD₆₀₀ was monitored every hour. Three or more independent experiments were performed for each bacterium. Nisin was added at the following concentrations: 2,500 IU/ml for Salmonella enterica, 250 IU/ml for Bacillus cereus, and 150 IU/ml for Staphylococcus aureus; the concentration of S-layer was 10 μg/ml.To determine the mode of action of the combination of S-layer and nisin, lysis was monitored by OD₆₀₀ decrease of exponential cultures of B. cereus and S. aureus when exposed to nisin or nisin plus S-layer and viable counts were determined by serial decimal dilutions on LB agar plates. A bacteriolytic effect was observed. As shown in Fig. ​Fig.2A,2A, B. cereus cultures lysed instantly in the presence of both compounds as observed from the decrease in OD and confirmed by the viable count determinations. However, S. aureus showed a 2-h lag period before lysis was observed (Fig. ​(Fig.2B).2B). This delay may be attributed to the typical cell aggregation profile characteristic of Staphylococcus cultures, which might hide the target for the S-layer and/or nisin. In fact, after this period cells disaggregated as visualized by microscopic observation (data not shown) and both compounds led to cell lysis. In addition, when viable counts were performed, a rapid decline in viability was observed even before the OD decrease. The reduction in viability after a 6-h treatment with both nisin and S-layer was 5 and 4 logs for B. cereus and S. aureus, respectively.Open in a separate windowFIG. 2.Type of effect of the combination of nisin and S-layer. Exponentially growing bacteria were incubated in the presence of either nisin or nisin and S-layer. (A) Effect on Bacillus cereus with nisin (250 IU/ml) or nisin and S-layer (250 IU/ml and 10 μg/ml, respectively). (B) Effect on Staphylococcus aureus with nisin (150 IU/ml) or nisin and S-layer (150 IU/ml and 10 μg/ml, respectively). Dotted line, cfu/ml; unbroken line, OD at 600 nm.Concerning the Gram-negative Salmonella enterica, a peculiar behavior was observed: while whole cells suspended in buffer were lysed by the sole presence of S-layer (17), no lytic effect was observed when exponentially growing cultures were treated even with both S-layer and nisin (data not shown). However, the S-layer protein remains intact after incubation with whole cells as observed by Western blotting after 8 h of incubation (Fig. ​(Fig.3).3). We suspect that actively dividing cells of this Gram-negative pathogen may release an inhibitor either of the S-layer activity or of nisin. Further experiments should be performed in order to evaluate these hypotheses.Open in a separate windowFIG. 3.S-layer integrity during incubation with Salmonella. Viable cells from Salmonella enterica were washed once with phosphate-buffered saline buffer, resuspended at an optical density at 600 nm of 1 mixed with S-layer protein (10 μg/ml), and incubated at 37°C with constant rotation to keep the contents in suspension at the indicated times. At different times S-layer was analyzed by Western blotting. Electrotransfer to parablot polyvinylidene difluoride was according to the manufacturer''s instructions (Macherey-Nagel, Germany). Polyclonal antibody anti-S-layer was used at 1:1,000.To check if the synergic effect of S-layer on nisin is indeed due to its murein hydrolase activity, a mild condition of heat inactivation (60°C for 2 h) was assayed with the S-layer. This treatment inactivates the endopeptidase activity as seen by zymogram analysis (Fig. ​(Fig.4,4, zymogram, lane 3) but conserves the protein without dramatic changes, since no differences in migration or antibody detection were observed (compare lanes 3 and 1 in Fig. ​Fig.4).4). The addition of mild-heat-inactivated S-layer protein and nisin to S. aureus cultures was unable to inhibit growth (Table ​(Table1).1). In contrast, while nisin and S-layer separately inhibited growth to 22% or 0%, respectively, the addition of both inhibited dramatically the growth (89%) and growth rate (90%), representing an 8-log reduction in viable cell counts, pointing to the benefit of this synergetic effect (Table ​(Table11).Open in a separate windowFIG. 4.S-layer treatments. S-layer preparations were submitted to different treatments and analyzed by PAGE, zymography, and Western immunoblotting as previously described (16). Lane 1, S-layer; lane 2, pretreatment with nisin of L. acidophilus cultures before S-layer preparation; lane 3, heat-inactivated S-layer (2 h at 60°C); lane 4, S-layer preparation incubated with nisin.

TABLE 1.

Nature of the synergic combination^a

Proliferative activity in the cerebellar external granular layer evaluated by bromodeoxyuridine labeling

We have optimised an indirect immunoperoxidase technique demonstrating bromodeoxyuridine (BrdU) incorporation into dividing cells for cerebellar tissue sections of four-day-old rats injected with this marker. This permits confident identification of granule-cell precursors engaged in DNA synthesis in the external granular layer of the developing cerebellum. Preservation of BrdU immunoreactivity is attained using methanol/acetic acid fixation and different pretreatments before immunostaining, while unlabeled nuclei can be recognized clearly after Feulgen or hematoxylin counterstaining. We established conditions to ensure satisfactory BrdU uptake without affecting cell-cycle progression during the postlabeling time period. The dose of BrdU employed provides saturation S-phase labeling from at least 1 h after BrdU delivery. Various kinetic parameters and phase durations have been determined in experiments involving a single injection or cumulative labeling sequences, and the cycle time was calculated based on two models of generative behavior: steady-state and exponential growth. The working hypothesis of steadystate kinetics can be adopted successfully if the existence of neuroblasts with different proliferation rates is taken into account.     

Cell cycle -dependent proteolysis in plants. Identification Of the destruction box pathway and metaphase arrest produced by the proteasome inhibitor mg132

It is widely assumed that mitotic cyclins are rapidly degraded during anaphase, leading to the inactivation of the cell cycle-dependent protein kinase Cdc2 and allowing exit from mitosis. The proteolysis of mitotic cyclins is ubiquitin/26S proteasome mediated and requires the presence of the destruction box motif at the N terminus of the proteins. As a first attempt to study cyclin proteolysis during the plant cell cycle, we investigated the stability of fusion proteins in which the N-terminal domains of an A-type and a B-type tobacco mitotic cyclin were fused in frame with the chloramphenicol acetyltransferase (CAT ) reporter gene and constitutively expressed in transformed tobacco BY2 cells. For both cyclin types, the N-terminal domains led the chimeric cyclin-CAT fusion proteins to oscillate in a cell cycle-specific manner. Mutations within the destruction box abolished cell cycle-specific proteolysis. Although both fusion proteins were degraded after metaphase, cyclin A-CAT proteolysis was turned off during S phase, whereas that of cyclin B-CAT was turned off only during the late G2 phase. Thus, we demonstrated that mitotic cyclins in plants are subjected to post-translational control (e.g., proteolysis). Moreover, we showed that the proteasome inhibitor MG132 blocks BY2 cells during metaphase in a reversible way. During this mitotic arrest, both cyclin-CAT fusion proteins remained stable.     

Role of calcium in signal transduction during the hypersensitive response caused by basidiospore-derived infection of the cowpea rust fungus

The hypersensitive response (HR) of disease-resistant plant cells to fungal invasion is a rapid cell death that has some features in common with programmed cell death (apoptosis) in animals. We investigated the role of cytosolic free calcium ([Ca2+]i) in the HR of cowpea to the cowpea rust fungus. By using confocal laser scanning microscopy in conjunction with a calcium reporter dye, we found a slow, prolonged elevation of [Ca2+]i in epidermal cells of resistant but not susceptible plants as the fungus grew through the cell wall. [Ca2+]i levels declined to normal levels as the fungus entered and grew within the cell lumen. This elevation was related to the stage of fungal growth and not to the speed of initiation of subsequent cell death. Elevated [Ca2+]i levels also represent the first sign of the HR detectable in this cowpea-cowpea rust fungus system. The increase in [Ca2+]i was prevented by calcium channnel inhibitors. This effect was consistent with pharmacological tests in which these inhibitors delayed the HR. The data suggest that elevation of [Ca2+]i is involved in signal transduction leading to the HR during rust fungal infection.     

Mendelian randomization supports causality between maternal hyperglycemia and epigenetic regulation of leptin gene in newborns

Leptin is an adipokine that acts in the central nervous system and regulates energy balance. Animal models and human observational studies have suggested that leptin surge in the perinatal period has a critical role in programming long-term risk of obesity. In utero exposure to maternal hyperglycemia has been associated with increased risk of obesity later in life. Epigenetic mechanisms are suspected to be involved in fetal programming of long term metabolic diseases. We investigated whether DNA methylation levels near LEP locus mediate the relation between maternal glycemia and neonatal leptin levels using the 2-step epigenetic Mendelian randomization approach. We used data and samples from up to 485 mother-child dyads from Gen3G, a large prospective population-based cohort. First, we built a genetic risk score to capture maternal glycemia based on 10 known glycemic genetic variants (GRS₁₀) and showed it was an adequate instrumental variable (β = 0.046 mmol/L of maternal fasting glucose per additional risk allele; SE = 0.007; P = 7.8 × 10⁻¹¹; N = 467). A higher GRS₁₀ was associated with lower methylation levels at cg12083122 located near LEP (β = −0.072 unit per additional risk allele; SE = 0.04; P = 0.05; N = 166). Direction and effect size of association between the instrumental variable GRS₁₀ and methylation at cg12083122 were consistent with the negative association we observed using measured maternal glycemia. Lower DNA methylation levels at cg12083122 were associated with higher cord blood leptin levels (β = −0.17 log of cord blood leptin per unit; SE = 0.07; P = 0.01; N = 170). Our study supports that maternal glycemia is part of causal pathways influencing offspring leptin epigenetic regulation.